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Background  Urine citrate is reabsorbed exclusively along the renal proximal tubule  via  the apical Na + -dicarboxylate cotransporter NaDC-1. We previously showed that an acid load  in vivo  and media acidification  in vitro  increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ET B ) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.   Methods  We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ET B  and measured [ 14 C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2 +/+ ) and Pyk2-null (Pyk2 −/− ) mice were exposed to NH 4 Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.   Results  Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1  in vitro . Consistent with these results, NH 4 Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2 −/−  mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ET B  stimulation of NaDC-1  in vitro . Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.   Conclusions  Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ET B  in this process.

Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways